Significance of the diluent components

SIGNIFICANCE OF THE DILUENT COMPONENTS

(Tris, Fructose, Glycerol, Antibiotics, Importance of egg yolk as medium and Preparation of egg yolk)

Tris (Tris hydroxyl methyl aminomethane)

  • Tris is commonly called as universal diluent which prolongs the life of sperm at room temperature, penetrate sperm and acts as intra cellular buffer, less toxic at the critical low freezing temperature.

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Fructose

  • Fructose provides the glycolisable substrate to the sperm, prevents sperm agglutination, maintains required osmotic tension, provides electrolyte balance and added cryoprotection during freezing.  
Fructose added in the diluent is the major energy source for the sperm.

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Glycerol 

Glycerol - It is an intra cellular cryoprotectant which brings its action by penetrating the sperm cells.

Glycerol is the most widely used cryoprotective agent for cattle and buffalo bull spermatozoa. It was the spectacular discovery of Polge in 1949 that showed that the death of spermatozoa could be avoided if the cells were suspended in a medium containing glycerol 

The cryoprotective effect of glycerol was found by Polge (1949).

It act as cryoprotective agent by

  • Modifying the shape and size of the ice crystals formed
  • Binds with water and decreases freezing point of solution and less ice formed.
  • It acts through salt buffering mechanism
  • Reduces solute concentration
  • Prevents denaturation of proteins and rupture of plasma membrane of sperm.

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Antibiotics

  • Many of the organisms present in the semen are non-pathogenic.
  • The organisms which are contagious spreaded through the semen are Brucella abortus, Vibrio fetus, Trichomonos fetus, leptospira Pomona, Mycobacterium tuberculosis, mycobacterium paratuberculosis and viral agents like infectious bovine rhinotracheitis and FMD.
  • Refrigeration greatly suppresses the multiplication of organisms but does not necessarily stop it.
  • Some of the organisms like Pseudomonos aeroginosa, Trichomonas fetus, Leptospira Pomona, FMD and IBRT were surviving after freezing also.
  • Penicillin and streptomycin are two antibiotics widely used since 1950 and are still in use. They are relatively harmless to sperm cells and inhibit broad spectrum microorganisms.
  • Penicillin G – 500-1000 IU/ml and dihydrostreptomycin 500-1000 µg/ml is adequate for routine use.
  • Antibiotics will not completely eliminate Brucella abortus, Trichomonos fetus, Mycobacterium tuberculosis, Mycobacterium paratuberculosis, viral agents like infectious bovine rhinotracheitis and foot and mouth virus and Rickettsiae and Fungi.
  • Hence the addition of antibiotic is not an extra precaution and cannot be taken as effective tool to permit use of semen from infected bulls or unhygienic proceedings.

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Importance of Egg Yolk as a Medium

  • The value of the yolk of the hen’s egg as a diluting medium in semen preservation was found by Philips during 1939. 
The beneficial effect of egg yolk in semen freezing was found by Philips (1939).

  • It helped in rapid growth in recent years of artificial insemination throughout the world. Egg yolk protects lipoprotein sheath of sperm against cold shock.
  • The active principle in yolk responsible for this is now thought to be lecithin or a similar phospholipid occurring either free or in combination with protein. 
Lecithin - It is a component of egg yolk which prevents the cold shock to the sperm during feezing of semen.

  • It contains glucose, proteins, vitamins and required viscosity index which may be of advantage to the cells.
  • The yolk contains the amino on oxidative deamination.
  • The hydrogen peroxide formed, which is toxic to the sperm, may be destroyed by additions of the enzyme catalase to the yolk diluents, under anaerobic conditions the problem does not occur.

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Preparation of Egg Yolk

  • Unfertilized egg is used for this purpose.
  • The egg should be purchase from disease free, known flock.
  • Fresh egg immediately after laying is purchased
  • The egg immediately after receiving is wiped with dry cotton and stored at refrigerator at 4-5⁰C. 
The egg should be cleaned with dry cotton only. Don't use water to clean the egg. If the egg is cleaned with water, the pores on the surface of the egg shell will be opened and the microorganisms will enter inside.

  • Maximum it can be stored for 3 days at refrigerator temperature of 4-5⁰C
  • Immediately before use it is cleaned with swab of 70% alcohol
  • It is opened at the narrow end with the help of a sterilized forceps.
  • The white of an egg (albumen) is drained off with the help of an egg yolk separator and yolk is placed on a sterile filter paper.
  • If required yolk may be transferred to another filter paper to clear traces of albumin.
  • Then the vitelline membrane is punctured with a sterile glass rod and yolk is allowed to fall in a sterilized graduated cylinder directly at the bottom without touching the walls.
  • The required quantity is prepared and added with autoclaved buffer (added with glycerol) and mixed with magnetic stirrer.

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Last modified: Tuesday, 12 June 2012, 4:07 AM